Control of protein synthesis through mRNA pseudouridylation by dyskerin

Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although pseudouridylation is the most abundant, its biological role remains poorly understood. Here, we demonstrate that the pseudouridine synthase dyskerin associates with RNA polymerase II, binds to thousands of mRNAs, and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a guide RNA with full complementarity. In cells lacking dyskerin, mRNA pseudouridylation is reduced, while at the same time, de novo protein synthesis is enhanced, indicating that this modification interferes with translation. Accordingly, mRNAs with fewer pseudouridines due to knockdown of dyskerin are translated more efficiently. Moreover, mRNA pseudouridylation is severely reduced in patients with dyskeratosis congenita caused by inherited mutations in the gene encoding dyskerin (i.e., DKC1). Our findings demonstrate that pseudouridylation by dyskerin modulates mRNA translatability, with important implications for both normal development and disease.

. Detection of dyskerin in nuclear speckles employing different antibodies and cell types (A) U2OS cells immunostained for dyskerin (green, Abnova antibody) and nucleolin (marker of nucleoli; red) or coilin (marker of Cajal bodies; red). Intensity profiles were calculated across the nucleolus and Cajal body indicated between the two arrows. Scale bar (white line) = 10µm. (B) U2OS cells immunostained for dyskerin (green, Atlas antibody) and the speckle protein SRRM2 (red). PFA fixation indicates fixation with 4% formaldehyde followed by permeabilization; 'Pre-extraction' indicates permeabilization of cells with cytoskeleton buffer 5 minutes prior to fixation with PFA; 'RNase A' is the same as 'pre-extraction' but with addition of 300 μg/ml RNase A to the cytoskeleton buffer. The dyskerin antibody used here is a second independent antibody raised in another species compared to the one used in Figure  1A. Scale bar = 10µm. (C) WI-38 fibroblasts pre-extracted as above and immunostained for dyskerin (green, Abnova antibody) and SRMM2 (red). Scale bar = 10µm.
(D) U2OS cells were treated with RNase A and immunostained using antibodies for GAR1, NHP2 or NOP1 (green) and the speckle marker SC-35 (red). Intensity profiles were calculated above the speckle(s) indicated between the two arrows. Scale bar = 10µm. (E) U2OS cells were subjected to siRNA treatment for 48 hours and then analyzed by qPCR for expression of the mRNA indicated. RNA levels were normalized to those of beta-actin mRNA and are shown relative to the siControl sample (mean ± SD, n=3 independent experiments). **** p ≤ 0.0001, as determined by a paired ratio two-tailed t-test.

Fig. S2. Dyskerin binds to genes through RNAPII in different cell lines (A)
ChIP-qPCR results showing dyskerin and RNAPII (precipitated with the 8WG16 antibody) enrichment at the ACTB locus in U2OS cells treated or not with triptolide for 2 hours. The PCR-amplified regions inside the ACTB gene are indicated with green lines in the scheme. Data are represented as mean ± SD, n=3 independent experiments. * p<0.05, *** p<0.001, **** p<0.0001, as determined by unpaired, two-tailed Student's t-test. (B) Immunoprecipitation of RNAPII or IgG from the chromatin fraction of U2OS cells with or without RNase A treatment followed by western blotting of the proteins indicated. A characteristic blot is shown. (C) Heatmap showing the correlations between dyskerin ChIP-seq binding profiles in U2OS and HCT116 cells. The numbers shown represent Spearman's correlation coefficient. (D) Shown is an example of the ChIP-seq profile of dyskerin and RNAPII at the RPL18A gene (i.e., the host gene for SNORA68) in U2OS cells. The ChIP-seq signal is normalized to input, and averaged across of 2 independent experiments. (E) Cumulative distribution of genes that contain at least one snoRNA or scaRNA inside their introns across genes ranked according to the normalized dyskerin binding in ChIP-seq (blue) or RNA-seq expression (TPM; red). Dyskerin ChIP binding and RNA expression were ranked from highest to lowest from left to right. (B) MA plot of the differentially expressed genes after dyskerin and GAR1 knockdown (as determined by DESeq2) in the RNA-seq samples described above. Genes were considered differentially expressed if their absolute log2 fold change over control was higher than 1. (C) Graph showing the number of transcripts deregulated by dyskerin or GAR1 knockdown (KD) in the RNA-seq samples described above. The dots indicate the sets to which the bars refer to, while the lines indicate the intersections between the sets. (D) Pie chart of RNA differentially expressed after dyskerin or GAR1 knockdown in the RNAseq samples described above, organized by RNA biotype group. (E) Percentage of significant splicing changes in cells after dyskerin (top) or GAR1 (bottom) knockdown in the RNA-seq samples described above compared to control cells, divided by the type of alternative splicing. The numbers indicate the number of different splicing events.  The graph shows that binding of dyskerin and GAR1 to repeat elements is specific, and not due to generic association of the two proteins with intronic sequences. (E) Distribution of dyskerin and GAR1 iCLIP peaks among different classes of repeat elements. (F) Cumulative distribution functions of protein-coding RNAs bound by dyskerin (blue) and GAR1 (red) across protein-coding genes ranked by dyskerin ChIP-seq binding. Dyskerin ChIPseq binding was ranked from highest to lowest from left to right. (G) Venn diagram showing distribution of binding of dyskerin or GAR1 to protein-coding RNAs (blue circles), sno/scaRNAs (red circles) and LINE/Alu RNA (yellow circles). The intersections represent sno/scaRNAs or LINE/Alu that are encoded from introns of expressed protein-coding mRNA bound by dyskerin or GAR1.
(H) Distribution of iCLIP reads from dyskerin and GAR1, divided by RNA biotype group, and normalized by their length and expression. Shown is the average of two independent experiments.

Fig. S5. mRNAs bind dyskerin and are pseudouridylated in fibroblasts and lymphoblasts (A)
GFP mRNAs containing only uridine (0% pseudouridine) or pseudouridine (100%) were subjected to RIP with an antibody against pseudouridine or IgG to verify its specificity. The graph shows the amount of co-precipitated RNA as percentage of input (mean ± SD, n=3) measured by qPCR. * p<0.05, ns non-significant, as determined by unpaired, two-tailed Student's t-test. (B) Chromatin fractions of U2OS cells treated with the siRNA indicated for 48 hours were subjected to RNA immunoprecipitation using an antibody targeting pseudouridine. 50 ng of in vitro-transcribed GFP mRNA with 100% pseudouridine content were added per 100 μg of protein lysates prior to addition of antibody. The graph shows the amount of co-precipitated RNA (mean ± SD, n=3 independent experiments) measured by qPCR and presented as percentage of input relative to siControl. *** p<0.001, **** p ≤ 0.0001, as determined by twotailed paired ratio t-test. (C) Chromatin fractions from primary fibroblasts (left) and immortalized lymphoblasts (right) from healthy donors were subjected to RNA immunoprecipitation using an antibody targeting dyskerin or a negative IgG control. The graph shows the amount of co-precipitated RNA as percentage of input (mean ± SD, n=3 independent experiments) measured by qPCR. * p<0.05, ** p<0.01, ns non-significant, as determined by two-tailed Student's t-test. (D) Chromatin fractions from primary fibroblasts (left) and immortalized lymphoblast (right) from healthy donors were subjected to RNA immunoprecipitation using an antibody targeting pseudouridine or a negative IgG control. The graph shows the amount of co-precipitated RNA as percentage of input (mean ± SD, n=3 independent experiments) measured by qPCR. * p<0.05, ** p<0.01, as determined by two-tailed Student's t-test.

Fig. S6. Elevated expression of exogenous mRNAs in dyskerin-depleted cells (A)
U2OS 2-6-3 CLTon cells were treated for 48 hours with siRNA and for the last 3 hours with doxycycline to induce the expression of the exogenous rabbit beta-globin cassette and then analyzed by qPCR for expression of rabbit beta-globin. The RNA levels were normalized to those of beta-actin mRNA and are shown relative to the control sample (mean ± SD, n=3 independent experiments). * p<0.05, as determined by two-tailed paired-ratio t-test. (B) U2OS cells were transfected with siRNA for 48 hours and for the last 6 hours with a plasmid encoding GFP or mCherry and then analyzed by qPCR for expression of GFP or mCherry. The RNA levels were normalized to those of beta-actin mRNA and are shown relative to the control sample (mean ± SD, n=3 independent experiments). * p<0.05, ** p<0.01, as determined by two-tailed paired ratio t-test. (B) MCF7, HCT116 and WI-38 cells were subjected to siRNA treatment for 48 hours, pulsed with puromycin for 10 minutes followed by 30 minutes recovery and western blotting. Shown are representative blots. (C) U2OS cells were transfected with siRNA for 48 hours, pulsed with puromycin for 10 minutes followed by 30 minutes recovery, and extracted for western blotting. Shown are a representative blot and the densitometric quantification of puromycin (normalized to β-actin) and p-eIF2⍺ S51 (normalized to β-actin and total eIF2⍺) levels relative to the levels of siControl (mean ± SD, n=3 biological replicates). ** p<0.01, as determined by two-tailed paired ratio ttest. The puromycin blot is the same as the one shown in Figure 6E. (D) U2OS cells were transfected with siRNA for 96 hours, pulsed with puromycin for 10 minutes followed by 30 minutes recovery and extracted for western blotting. Shown are a representative blot and the densitometric quantification of puromycin (normalized to β-actin) and p-eIF2⍺ S51 (normalized to β-actin and total eIF2⍺) levels relative to the levels of siControl (mean ± SD, n=3 biological replicates). The puromycin blot is the same as the one shown in Figure 6E. p values as determined by two-tailed paired ratio t-test. (E) Fibroblasts (top) and lymphoblasts (bottom) from patients with dyskeratosis congenita (DC) and healthy donors were pulsed with puromycin for 10 minutes followed by 30 minutes recovery and extracted for western blotting. Shown are a representative blot and the densitometric quantification of puromycin and dyskerin normalized to β-actin and relative to the levels of healthy control (mean ± SD, n=3 independent experiments). * p<0.05, ** p<0.01, as determined by two-tailed paired ratio t-test. (F) U2OS cells were transfected with siRNA for 48 hours and for the last 16 hours with a plasmid encoding GFP or mCherry followed by western blotting for the proteins indicated.